The control of mRNA stability is a critical determinant in the post-transcriptional regulation of eukaryotic gene expression. Even minor alterations in mRNA stability can have profound consequences and may manifest as clinical phenotypes as illustrated by the ability of aberrantly expressed proto-oncogenes that can give rise to malignancies. Despite the importance of mRNA stability in the control of gene expression, progress has only recently been made in the identification and characterization of the components that control mRNA turnover and their ultimate physiological consequence. We have focused our efforts on the study of nucleases involved in mRNA decay, in particular, mRNA 5'end decapping and the cell biological significance of decapping. We have now identified multiple confirmed and putative decapping enzymes, which appear to modulate a select subset of mRNAs and pathways. In this proposal we will expand on the cell biological and functional role of mRNA decapping enzymes. We have shown the Dcp2 decapping enzyme selectively modulates the decapping of mRNAs involved in innate immunity and will pursue its role in the innate immune response in Aim1. We have identified a novel class of decapping proteins represented by the mammalian DXO protein, which possesses an unusual intrinsic dual decapping and exonuclease activities. We have already shown DXO preferentially functions on incompletely capped pre-mRNAs in a nuclear pre-mRNA 5'end quality control mechanism, and now have evidence it also functions as a canonical decapping enzyme disproportionally modulating a subset of mRNAs involved in cytoskeletal architecture and cell migration. We will pursue the role of DXO in mRNA decapping and cell migration in Aim2. In Aim3, we will build on our ongoing efforts to identify additional mRNA decapping enzymes and decipher the functional role of six new Nudix family proteins we recently demonstrated contain decapping activity in vitro, Nudt2, Nudt3, Nudt12, Nudt15, Nudt17 and Nudt19. We will initially focus on the evolutionarily conserved Nudt3 protein where preliminary data indicates it is a bona fide decapping enzyme in cells. Collectively, this work will provide novel insights into a fundamental post-transcriptional regulatory mechanism involved in gene expression and a framework for innovative approaches for therapeutic intervention in pathological conditions.